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Veterinary Parasitology 153 (2008) 100–107

The ecology of horse cyathostomin infective larvae

(Nematoda-Cyathostominae) in tropical southeast Brazil

Simone Quinelato a, Melissa C.M. Couto a, Bruno C. Ribeiro b,Claudia N. Santos b, Luciene S. de Souza b, Debora H.S. dos Anjos c,

Ivan B.M. Sampaio d, Lurdes M.A. Rodrigues e,*a UFRRJ, Postgraduate Program in Veterinary Sciences, Dept. of Animal Parasitology, BR 465, Km 7, 23890-000, Seropedica, RJ, Brazil

b UFRRJ, Scientific Initiation CNPq/PIBIC, Dept. of Animal Parasitology, BR 465, Km 7, 23890-000, Seropedica, RJ, Brazilc UFRJ, Carlos Chagas Filho Biophysics Institute, CCS-BI, 21949-900, Rio de Janeiro, RJ, Brazil

d UFMG, Veterinary School, Zootechny Dept., Av. Antonio Carlos, 6627, 31270-901 Pampulha, MG, Brazile UFRRJ, IV, Dept. of Animal Parasitology, CPGCV, BR 465, Km 7, 23890-000 Seropedica, RJ, Brazil

Received 24 August 2007; received in revised form 9 January 2008; accepted 11 January 2008

Abstract

Experimental studies about the recovery, survival and migration to pasture of cyathostomin infective larvae (L3) from fresh feces

depositions were conducted from February 2005 to March 2007 in a tropical region of southeast Brazil. Grass and feces were

collected weekly at 8 a.m., 1 and 5 p.m. and processed by the Baermann technique. Multivariate analysis (principal components

method) showed the influence of time and environmental variables on the number of infective larvae recovered from the feces and

pasture. In the rainy period (October–March), more infective larvae were recovered on the feces and grass apex. In contrast, in the

dry period (April–September), the recovery was higher only on the grass base, as well as the L3 survival on feces and grass. More

larvae were recovered at 8 a.m., except from the grass apex, where the highest recovery was at 1 p.m. Few studies investigating the

seasonal transmission of equine cyathostomin have been conducted in South American tropical climates. These results demonstrate

that in tropical conditions L3 are available on feces and pasture throughout the year. Knowledge of climatic influences on the

development and survival of L3 is crucial to designing integrated parasite control programs that provide effective protection while

slowing the development of anthelmintic resistance.

# 2008 Elsevier B.V. All rights reserved.

Keywords: Cyathostomin; Horses; Tifton 85; Infective larvae

1. Introduction

Gastrointestinal nematodosis is an important disease

among grazing horses in all regions of the world (Love

and Duncan, 1992). Control programs are based on the

intensive use of drugs, promoting the development of

* Corresponding author.

E-mail addresses: squinelato@ufrrj.br (S. Quinelato),

lurdesar@ufrrj.br (L.M.A. Rodrigues).

0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2008.01.027

nematode resistance to most anthelminthics groups.

Alternative methods to control cyathostomin infection

should take into account the infective larvae dynamics

on pasture (Ramsey et al., 2004; Nielsen et al., 2007).

It has been proposed that the ‘‘parasite refugium’’ is

the most important factor in the development of

nematode resistance (van Wyk, 2001; Kaplan, 2004).

This refugium is constituted by free-living stages in

pasture and untreated animals and some encysted

parasites, such as the histotrophyc phase of the

cyathostomin (Nielsen et al., 2007).

S. Quinelato et al. / Veterinary Parasitology 153 (2008) 100–107 101

The development, survival and migration of infective

larvae are influenced by environmental conditions, such

as temperature and rainfall. Larvae migration is

regulated by available moisture and survival is

influenced strongly by temperature (Ogbourne, 1973;

Craig et al., 1983; Eysker et al., 1986; Quinelato et al.,

2007).

Some types of grass maybe favor L3 survival and

migration. Studies of the development and survival of

cyathostomin eggs and larvae in pastures have been

carried in temperate climate areas (Ogbourne, 1972;

Duncan, 1974), however few such studies have been

conducted in tropical areas (Mfitilodze and Hutchinson,

1988; Hutchinson et al., 1989; Quinelato et al., 2007)

and subtropical ones (English, 1979a,b; Craig et al.,

1983; Courtney and Asquith, 1985; Baudena et al.,

2000a,b).

This effort is essential to South American tropical

region, since there are only a few studies related to these

conditions. The objective of this study is to evaluate the

recovery, survival and migration of cyathostomin

infective larvae on Tifton 85 grass (Cynodon spp. cv.

Tifton 85) at different times of the day during 24

months. Such knowledge will contribute to control

horse infection while reducing the use of anthelmintics.

2. Material and methods

2.1. Experimental design

From February 2005 to March 2007 the potential

transmission of infective larvae (L3) of equine

cyathostomin was studied, employing the methodology

described in Quinelato et al. (2007). The samples of

feces and grass were collected at three times of the day

(8 a.m., 1 p.m. and 5 p.m.). The grass was divided into

base (0–20 cm) and apex (20–40 cm). For data analysis,

two different periods were considered: rainy (October–

March) and dry (April–September).

2.2. Location and meteorological data

The study was carried out at the Helminthology

Laboratory of W. O. Neitz Parasitology Station,

UFRRJ, Rio de Janeiro, Brazil. The climate is AW

type (tropical humid), according to the Kopen

classification.

The weather data were supplied by the Seropedica

Agricultural Weather Station, INMET/PESAGRO, RJ.

The soil temperature was measured at the experi-

mental area with a soil thermometer at each time of

collection.

2.3. Animals and pasture

Fecal pats were obtained from horses naturally

infected with cyathostomin. These animals did not

receive any anthelminthic treatment during experi-

mental period. ATifton 85 plot (Cynodon spp. cv.Tifton

85) was set off measuring 11.50 m � 1.00 m, fenced to

prevent the animals from entering and grazing.

2.4. Fecal pats

A fecal pat of approximately 1 kg was collected

directly from the horse, the number of eggs per gram

(epg) was processed by McMaster technique (Gordon

and Whitlock, 1939) and the larvae were cultured

according to Roberts and O’Sullivan (1950). After these

procedures, the feces were placed in the pasture plot in

the first week of each month, separated by �50 cm.

2.5. Procedures

Seven days after the deposit, one sample of feces

(�2 g) and another of grass were collected weekly from

three different field sites. Collection of feces and grass

were done at three different times a day (8 a.m., 1 p.m.

and 5p.m.) from the feces deposited and the surrounding

grass plot. Grass sample was divided into base (0–

20 cm) and apex (20–40 cm). The samples were

weighed weekly and processed by the Baermann

technique (Ueno and Goncalves, 1998). After 24 h,

the L3 were recovered and observed under an optical

microscope. Infective larvae were counted and identi-

fied according to Bevilaqua et al. (1993). To obtain the

number of L3 kg�1, the feces and grass samples were

dried at 75 8C for 48 h.

The fecal plots were abandoned when no more L3

could be recovered from the feces and grass samples.

The data on larvae number in feces and grass,

survival period, average air and soil temperature and

total rainfall were recorded weekly in an Excel file.

2.6. Statistical analysis

To evaluate the effect of the three collection times,

the average values were compared by the Kruskal–

Wallis (P < 0.05) (Zar, 1999) nonparametric test

through the BioEstat program (Ayres et al., 2005).

Mann–Whitney nonparametric test (P < 0.05) (Zar,

1999) was used to analyze means values of temperature

(air and soil), rainfall, epg, infective larvae recovery and

survival in the rainy and dry periods, again through the

BioEstat program (Ayres et al., 2005).

S. Quinelato et al. / Veterinary Parasitology 153 (2008) 100–107102

The selected collections (n = 139) were submitted to

multivariate analysis (principal components method)

(Judez, 1989) using the INFOSTAT software. Larvae

counts within observation periods were set in percen-

tages in relation to total count of each period, in order to

properly observe migration dynamics. Graphical repre-

sentation of the variables is shown in a three-

dimensional system, allowing visualization of associa-

tions among them.

3. Results

3.1. Meteorological data

The average air and soil temperature and total

rainfall were high in the rainy period (P < 0.05)

(Fig. 1). In this period the average temperatures were

25.5 8C for air and 27.9 8C for soil, while in the dry

season these were 22.8 8C for air and 24.3 8C for soil.

The total rainfall was 141.93 mm and 56.55 mm in

rainy and dry period respectively.

Fig. 1. Monthly average air temperature (T), soil temperature (

Fig. 2. Number of eggs per gram (epg) fr

3.2. epg

epg levels varied during the experimental period,

with values between 850 and 4500 (Fig. 2). Peaks were

observed in December 2006 (rainy) and April and

August 2005 (dry). The average in the rainy period was

1823 (�269.49) and during the dry period the average

was 1925 (�346.92) (P > 0.05).

3.3. Feces

L3 recovery was observed in the first week, with larger

numbers in November 2006, (107,482 L3 kg�1 dm) and

April 2005 (106,695 L3 kg�1 dm). In the rainy period,

the average number of larvae recovered was

26,961 L3 kg�1 dm, and during the dry period it was

30,566 L3 dm (P > 0.05) (Fig. 3).

Infective larvae were recovered at three times of day

(8 a.m., 1 p.m. and 5 p.m.), with the largest recovery at 8

a.m. (P < 0.05), and with peaks in April 2005 and

November 2006 (Fig. 4).

Ts) and total rainfall from February 2005 to March 2007.

om February 2005 to January 2007.

S. Quinelato et al. / Veterinary Parasitology 153 (2008) 100–107 103

Fig. 3. Percentage of cyathostomin infective larvae recovered from fecal and grass samples from February 2005 to January 2007.

Larval survival varied from 2 to 9 weeks in the

rainy period and from three to 14 weeks in the dry

(Fig. 5), with highest L3 survival in the dry season

(P < 0.05).

3.4. Grass base

L3 migration in the rainy and dry seasons occurred

from the second week. The highest larval recoveries

were in August 2005 (3933 L3 kg�1 dm) and October

2005 (1728 L3 kg�1 dm). The average number of larvae

recovered was 555 L3 kg�1 dm in the rainy season and

762 L3 kg�1 dm in the dry (P < 0.05) (Fig. 3).

Fig. 4. Cyathostomin infective larvae recovery in feces at d

The L3 larval were collected at the three times

(P > 0.05), with the largest recovery at 8 a.m. and peaks

in August and October 2005 (Fig. 6).

The L3 survival varied from 1 to 9 weeks in the rainy

period and from 2 to 11 weeks in the dry (Fig. 5). There

was a difference in L3 survival between the periods

(P < 0.05), with the highest survival in the dry season.

3.5. Grass apex

The L3 migration occurred from the first week in the

rainy period and second week in the dry. The largest

number of L3 was recovered in December 2006

ifferent collection times (8 a.m., 1 p.m. and 5 p.m.).

S. Quinelato et al. / Veterinary Parasitology 153 (2008) 100–107104

Fig. 5. Cyathostomin infective larvae survival (weeks) in feces and grass samples from February 2005 to January 2007.

(919 L3 kg�1 dm) and June 2006 (335 L3 kg�1 dm).

The average number of larvae recovered were

263 L3 kg�1 dm (rainy) and 185 L3 kg�1 dm (dry)

(P > 0.05) (Fig. 3).

Just as for the other samples, the L3 cyathostomin

were recovered at three times of day, with the highest

recovery at 1 p.m. (P > 0.05) and a peak in October

2006 (Fig. 7).

The L3 survived up to 7 weeks in the rainy period and

10 weeks in the dry (P < 0.05) (Fig. 5).

Fig. 6. L3 recovery from grass base at different c

The factors identified in the multivariate analysis as

being associated with the number of L3 recovered from

the pasture samples included the time since the feces

were deposited, the average temperature (air and soil)

and the rainfall during the previous week. There was a

decrease in the percentage of L3 in feces (% lgf) and

grass (% lgg) as a function of time, with a great

difference observed in feces. Increasing rainfall

promoted an increase of the larval percentage in grass

(% lgg). Air temperature had a negative effect on L3

ollection times (8 a.m., 1 p.m. and 5 p.m.).

S. Quinelato et al. / Veterinary Parasitology 153 (2008) 100–107 105

Fig. 7. L3 recovery from grass apex at different collection times (8 a.m., 1 p.m. and 5 p.m.).

percentage in feces (% lgf), while soil temperature (tsoil)

negatively influenced negatively the L3 grass percen-

tage (% lgg) (Fig. 8).

4. Discussion

4.1. Feces

In tropical climates, horses can be infected at all

times of the year because L3 are present in the pasture

Fig. 8. Graphical representation of variables acting on infective larvae

pasture dynamics, according to axis 2 and 3. The coordinate and

direction of the first axis are represented by the arrow and its value.

System inertia: 72%. %lf, larvae in feces; %lg, larvae in grass; day,

days; rf, rainfall; tair, average of air temperature; tsoil, average soil

temperature.

throughout the year. The animals are more exposed to

infection in dry periods than in periods with intense

rains (Quinelato et al., 2007; Couto et al., personal

comment, 2007). If the quantify of forage is not enough,

animals can graze close to fecal pats, seeking more

palatable and new growth grass, with greater infection

risk (Craig, 1999).

In the present study, the larger L3 recovery in the dry

period occurred due to ideal environmental conditions

for larval development and migration to the pasture,

showing the importance of the fecal pats as an ideal

microclimate for egg and larval development and as

larvae reservoirs, as observed by Mfitilodze and

Hutchinson (1987), Uhlinger (1991), Quinelato et al.

(2007), Couto et al. personal comment (2007).

The higher number of L3 recovered at 8 a.m. was

probably associated to the mild air temperature and

fecal moisture in the morning (Hasslinger and Bittner,

1984).

According to the literature, factors that favor larval

development do not favor survival. High temperature

and moisture, typical of the rainy period, lead to fast

larvae development but limit their survival. In contrast,

in the dry period these climatic factors are milder and

larvae develop more slowly but survive longer

(Courtney, 1999; Nielsen et al., 2007).

The larger L3 survival observed in this study during

dry period can be explained also by the fact that pats

stay intact during this period, protecting the eggs and

larvae (Rupasinghe and Ogbourne, 1978). In the rainy

season, intense rains can destroy the fecal pats, reducing

the survival period. These results are in agreement with

S. Quinelato et al. / Veterinary Parasitology 153 (2008) 100–107106

English (1979a), who in studies in the Australian

subtropical area reported that development of eggs to L3

was completed in 2 weeks during the summer (rainy

period), but only 1–10% of the larvae survived. During

the winter (dry period), L3 development was completed

in 5 weeks, with 80% survival.

4.2. Grass

The larger L3 recovery in the grass base in the dry

period (total rainfall 56.55 mm), in contrast with the grass

apex in the rainy period (total rainfall 141.93 mm), can be

explained by the lower rainfall in the dry period, because

rainfall acts as limiting factor for larval migration to the

pasture (English, 1979b; Ogbourne, 1972, 1973; Hutch-

inson et al., 1989; Langrova et al., 2003). No correlation

was found by Hutchinson et al. (1989) between L3

recovered from pasture and the amount of rain. They

considered that rain just supplies the minimum moisture

necessary for migration and that great amounts of rain

would disperse L3. Most of the studies have analyzed

only L3 horizontal migration, preventing comparison

with our results. The larger recovery at 1 p.m. here does

not agree with the findings of Langrova et al. (2003),

where the larger values at 8 a.m. were explained by the

presence of morning dew.

Moisture influences larval migration, while tempera-

ture more directly influences survival, both in temperate

and tropical climates (Ogbourne, 1973; Craig et al., 1983;

Eysker et al., 1986). The larger L3 survival in the dry

period can be explained due to mild temperature and little

rain, allowing fecal pats to serve as an L3 reservoir. In

contrast, during the rainy season, the higher temperatures

raises larval metabolism, resulting in fast depletion of

their energy reserves, reducing the survival and

infectivity of the larvae (Cheah and Rajamanickam,

1997; Medica and Sukhdeo, 1997).

The type of grass can influence L3 recovery and

survival. The plants have different growth patterns and

wide morphologic contrasts, and these characteristics

alter the vegetation microclimate, directly affecting the

larval development and survival (Niezen et al., 1998).

Tifton 85 (Cynodon spp. cv. Tifton 85) is widely used

for equine grazing, it is a hybrid characterized by wide

leaves, few trichomes, big stems, fine stalks and well-

developed rhizomes (Burton et al., 1993). It grows low

to the ground, allowing wide solar light incidence on the

vegetation, which might have contributed to smaller

larval recovery in the hottest months (rainy period),

besides the drying of the fecal plot, hindering the

liberation of infective larvae. In the dry period, this

pasture type is more fibrous protecting the larvae from

unfavorable environmental conditions (Brady and Weil,

1999). In the present study, the highest number of

infective larvae was recovered from grass base, maybe

because the infective larvae find favorable conditions

for survival on this part of the grass and because the fine

stems provide smaller area for larval migration. Another

factor could be the presence of few trichomes, hindering

the formation of a moist layer that allows the access of

infective larvae to the upper parts of the grass.

The environmental conditions of the studied area are

favorable for the development of cyathostomin infective

larvae. The L3 are present in the pasture throughout of

the year, so methods of prevention and control are

necessary to help reduce the number of larvae,

especially those in refugia. Some methods of prevention

are simple and inexpensive, like monitoring feces epg

and pasture rotation. These can help to prevent infection

caused by larvae in refugia.

Another important point is the frequency of treatment

with anthelmintics. This frequency will depend on the

percentage of feces contamination and development of

anthelmintics resistance. It is important to know the time

between treatments and the expected reappearance of

eggs in feces. An optimum treatment interval must be

established considering some risk factors, such as pasture

type, grazing history, time of the year, stock density and

weather conditions (Jacobs et al., 1995).

5. Conclusion

At all collection times, the number of larvae

recovered was high with potential infection risk to

equines.

The L3 recovery was larger from the grass base

(0–20 cm) than apex (20–40 cm), with the highest L3

recovery from the base during the dry season. The largest

survival was observed in the dry period. The fecal pats

acted as reservoirs of the cyathostomin in both periods.

Knowledge of climatic influences on the develop-

ment and survival of free-living stages is essential for

the development of control programs that provide

efficient protection and still reduce the development of

anthelminthic resistance.

Acknowledgements

This study had financial support from CPGCV,

UFRRJ, CAPES and CNPq. We also so thank the

Seropedica Agricultural Weather Station (INMET/

PESAGRO) for providing the meteorological data used

in this study and Martin K. Nielsen for his research

contribution.

S. Quinelato et al. / Veterinary Parasitology 153 (2008) 100–107 107

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